Isoaspartate residues dramatically influence substrate recognition and turnover by proteases

Area of Science:

• Biochemistry
• Proteomics
• Enzymology

Background:

• Posttranslational modifications (PTMs) are crucial for protein function.
• Spontaneous PTMs, such as isoaspartate (isoAsp) formation, can alter protein structure and stability.
• IsoAsp formation may affect protein susceptibility to enzymatic degradation.

Purpose of the Study:

• To investigate the impact of isoaspartate formation on protease activity.
• To determine how different peptidases interact with isoAsp-containing substrates compared to Asp-containing substrates.

Main Methods:

• Enzyme assays using synthetic peptide substrates containing either aspartate (Asp) or isoaspartate (isoAsp).
• Testing a panel of proteases including prolyl oligopeptidase family members, caspases, amino peptidase N, matrix metalloproteinase 3 (MMP 3), enterokinase, acylamino acid-releasing enzyme, and dipeptidyl peptidases.

Main Results:

• Prolyl oligopeptidases, caspases, amino peptidase N, MMP 3, and enterokinase failed to cleave substrates containing isoAsp at specific positions.
• Acylamino acid-releasing enzyme and dipeptidyl peptidases 1, 2, and 4 successfully hydrolyzed substrates with an isoAsp-Ala motif.

Conclusions:

• Isoaspartate formation significantly alters protease substrate specificity.
• Certain peptidases are unable to process isoAsp residues, potentially influencing protein turnover and function in vivo.
• The study highlights the differential recognition of Asp and isoAsp by various enzyme classes.